A: The current definition of exosomes is complex, with no absolute consensus in the field. Typically, exosomes are defined as vesicles floating in sucrose solutions at a density ~1.13 to 1.19 g/ml during ultracentrifugation-based isolation, with an expected size of 30-150 nm (based on electron microscopy analysis). Exosomes can also be defined and identified by their surface protein markers, which include: tetraspanins (CD63, CD81, CD9) and others like ALIX. Currently, there are no appropriate tools nor sufficient knowledge in the field to set a clear and simple definition of exosomes that would differentiate them from other micro-/nanovesicles.

Q: What are exosomes composed of?

A: Exosomes are tiny vesicles (30-150 nm) containing protein and/or RNA cargo, within a lipid bi-layer membrane. Exosomes can differ extensively in both their cargo and surface proteins, and different cell types can secrete different - sometimes multiple - types of exosomes.

Q: What is the mechanism of exosome formation?

A: Exosomes are classically described as vesicles originating from the endocytic pathway through fusion of multivesicular bodies with the plasma membrane. They are a part of a larger family of vesicles secreted by cells - including microvesicles, ectosomes and shed particles - which originate by direct budding from the plasma membrane. It is extremely challenging to separate these entities using currently available techniques and instruments, due to overlap in their size, density and overall composition similarities.

Q: How do I know that I’ve isolated exosomes, and not other types of vesicles?

A: In addition to their 30-150 nm size, to be categorized as exosomes, the vesicles should be positive for certain surface protein markers such as tetraspanins. The most widely accepted marker is CD63, but CD81, CD9 are utilized as well. Western blotting for these targets on the sample of interest is a relatively simple way to confirm that the vesicles are indeed exosomes. However, the current definition of exosomes is not set in stone, as there is no absolute consensus in the field. It will probably take another several years to come up with the exact specification and nomenclature for all nano-/microvesicles (including exosomes).

Q: How are exosomes visualized and/or counted?

A: Exosomes are too small (30-150 nm) to be seen using a regular microscope, as this is limited to objects that are at least several micrometers in size. Structures as small as bacteria, and even very large viruses such as vaccinia virus (approx. 0.5µm in size), can be observed by light microscopy. Yet the resolution of the light microscope is too low for observing exosomes.

The typical methods of analysis for exosome size distribution include the NanoSight instrument and electron microscopy. Although very different in methodology, both technologies allow you to study nanoparticles down to 10 nanometers in size.

To determine the concentration of exosomes in the sample, you can use the NanoSight instrument or the Izon instrument. The NanoSight instrument enables counting and sizing of nanoparticles (10-1000 nm) using light scattering and browning motion, while the Izon instrument accomplishes the same thing using nanopore analysis.

Q: What is the best way to store my exosomes?

A: For short-term, exosomes can be stored at 4°C for up to 1 week. For long-term storage, exosomes can be stored at -20°C or -80°C. When storing exosomes long-term, it is important to consider if they will need to be thawed more than once. If multiple applications (and thus multiple thaws) is required for analysis, we recommend aliquoting the exosome suspensions into multiple tubes so that each tube will only undergo one freeze/thaw cycle. We have found that multiple freeze thaw cycles can cause damage to the exosomes and reduce their numbers.

Q: What should I do when my Westerns do not seem to work?

A: There are several typical reasons for this:

Not enough sample volume added. Exosomes can contain a fairly low amount of protein cargo. For an initial experiment, we recommend adding as much of the sample as possible

Antibodies are not optimal. We suggest testing antibodies (e.g. anti-CD63 or other exosomal marker) from 2-3 manufacturers, carefully checking what concentration is recommended. Also, they should ideally be used fresh, and need to be stored properly.

Depending on the exosomal surface marker, certain gel conditions might be more optimal for the target antibody (e.g. reducing/non-reducing and denaturing/non-denaturing). We suggest checking with the manufacturer and exosome community about which conditions are recommended for the specific marker you are targeting and the specific antibody you are using.

General Western techniques. Westerns can be tricky, and we recommend the use of a positive control for initial testing to make sure the entire workflow is functioning as it should. Any protein or antibody can be used as long as they meet the conditions you need (e.g. denaturing vs. non-denaturing). In addition, when picking the protein, try to steer clear of those that are present at very high or very low concentrations in your sample to prevent overloading the blot or total absence of signal.